The developmentally controlled amplification of ribosomal RNA genes (rDNA) in the ciliated protozoan Tetrahymena thermophila will be studied. The molecular steps involved in the pathway of amplification, which involves conversion of the single, chromosomally integrated gene at the rdn locus into free, linear, palindromic rDNA molecules, will be further defined. Mutants altered in rDNA amplification will be selected in order to dissect this pathway. Steps affected in the mutants, and in cells treated with inhibitors or different temperatures, will be determined by molecular analyses. DNA sequences at th rdn locus, or on two types of free rDNA molecules generated during amplification, will be compared between mutants and different wild type strains and related species, to identify regions of the gene important for amplification and its control. Using a mutant rDNA carrying a selectable drug resistance marker, methods will be developed for DNA-mediated gene transfer into Tetrahymena. This will enable the role of a putative intermediate of amplification to be tested directly. The choice of this system for studies of gene amplification is based on the feasibility of combining molecular with genetic experiments.
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